INTRODUCTION The centrality of oncogenes and tumor suppressor genes (TSG) in causation and development of human cancer provides the means to predict, detect, early diagnose and treat/cure human malignancies. The products of cancer causing genes are components of signal transduction circuits that control embryonic development, cellular proliferation and/or apoptotic death. Identification of these signaling pathways allows identifying molecular markers that herald the onset of steps in cancer development and predict clinical outcome or response to treatment. Cancer-causing genes are usually discovered in the inherited family cancer syndromes or through genetic mapping in tumor tissues or cell lines. The development of tumors from incipient malignant cells to metastases is driven by Darwinian expansion of clonal cell populations involving additional mutated cancer genes. Very recent discoveries in stem cell research are revolutionizing the field of cancer biology with tremendous implications for treatment strategies. It is becoming increasingly clear that a minute population of tumor stem cells exists within the tumor tissue. Therefore of paramount importance is the need to discover the lung cancer stem cells that constitute a small population of cells with self-renewal capacity essential for both tumor maintenance and spread. Fundamentally, these immortal tumor cells finally determine the response to treatment and tumor recurrence. Evidence from lung embryology and the morphology of combined lung tumors point to a common airway epithelial stem/progenitor cell of origin for most if not all epithelial lung tumors. Our research program began in 1987 and culminated in the identification in 1993 of the VHL TSG located at 3p25. In 1993-1999 we investigated the sequence structure of the VHL gene and identified VHL target genes. In parallel we intensified research on mapping and molecular cloning of TSGs located on 3p21.3 involved in the origin and /or development of major forms of lung cancer and carcinomas of the breast, ovary, cervix, testes, head and neck, prostate, and colon. In 2000-2003 we investigated: (1) the methylation code of the VHL locus itself and the function of VHL target genes (carbonic anhydrases, CA9 and CA12, and the transcription regulator STRA13); (2) continued deletion mapping in 3p21.3 by real time PCR in tumor tissues and cell lines; and (3) completed the isolation and initial characterization of candidate cancer-causing genes from both 3p21.3 regions (centromeric, LUCA, and telomeric, AP20). These 3p21.3 regions should be considered contiguous cancer gene regions harboring clusters of TSG. We then focused our research on functional analysis (finding interacting proteins, analyzing null mutants in mice, and bioinformatics annotations) of some of the strong candidate genes. VHL TSG (3p25) Using the VHL locus as a model system, we investigated the mechanisms of aberrant DNA methylation in cancer. We also aimed to use epigenetic codes (CpG and H3/H4 histone codes) to understand whether they harbor variations associated with inter-individual differences that may determine risk of sporadic cancers. We hypothesize that individual differences in epigenetic codes of TSG loci also manifested during normal aging may underlie the genetic predisposition to common cancers caused by the VHL gene. We produced detailed and contiguous "bisulfite-sequencing" profiles of the human and mouse VHL loci (17 and 6.7 kb respectively) to reveal CpG codes in addition to H3/H4 histone methylation profiles. In VHL-expressing cells, only the promoter CpG island is protected and free from methylation while the rest of the locus is heavily methylated. This methylation pattern was re-created de novo and maintained on the human genomic VHL transgene during development of transgenic mice, but not in transfected teratocarcinoma ES cells in culture. However, somatic cells were able to preserve the pre-existing methylation pattern after fusion in somatic cell hybrids and after human-to-mouse single chromosome 3 transfer. We will now focus on the identification of the protecting cis elements in the VHL locus and studies of the VHL epigenetic code variations. We discovered that CA 9 /CA12 genes are specifically induced and over-expressed in many tumor types and should be considered molecular markers of cancer growth and novel targets for treatment. These enzymes may control the compromised tumor microenvironment and should be considered molecular targets for development of new treatment modalities (cDNA based vaccines and new CA inhibitors). Using purified CAIX/XII enzymes we are testing novel CA inhibitors that may have potent anti-tumor activity (they also could be used to treat glaucoma). The STRA13 transcription factor is negatively controlled by the VHL gene and induced by hypoxia, a condition prevalent in cancer tissues. We established by Northern analysis and immunostaining very high expression levels of this gene in many human tumors but not in surrounding and matching normal tissues. We discovered that STRA13 binds to and modulates the activity of the powerful regulator of transcription STAT3 that is also activated in many cancers suggesting that STRA13 may be involved in regulating cancer cell growth and survival. Because the STAT3 transcriptional system is required for embryonal stem cell potency and self-renewal it would be necessary to investigate experimentally the expression of Stra13 in mESC. The 3p21.3 TSG We used the yeast two-hybrid system, controlled expression of trangenes in tumor cells, and bioinformatics to discover the function of the resident putative TSG. We identified the RASSF1A gene as a multiple TSG involved in many tumors, including lung, breast, prostate, kidney, head & neck, uterine cervix and others. We hypothesize that RASSF1 genes and their paralogs are inactivated in approximately 70% of human cancers. The HYAL2 protein was identified as a GPI-anchored receptor for the sheep lung cancer retrovirus, JSRV, and a sequestration mechanism inactivating HYAL2 protein was demonstrated. The env gene of JSRV was shown to transform human bronchial epithelial cells in vitro and sequester the HYAL2 protein. The absence of HYAL2 (mediated either by a putative virus or mutational inactivation) leads to ligand-independent activation of the RON receptor tyrosine kinase and its downstream signaling pathways (Akt and MAPK). We have been also studying the involvement of RON in SCLC. We discovered that in SCLC the promoter of RON is silenced by hypermathylation leading to simultaneous activation of a putative internal promoter. The novel transcript originating from this internal promoter encodes mostly the cytoplasmic portion of the receptor that is constitutively activated and may function as a proto-oncogene. RON therefore is emerging as a potential oncogenic factor in lung cancer and a target for therapeutic intervention. We discovered that the PL6 protein binds to and modulates the function of two similar progesterone receptors (MAPRs) localized in the endoplasmic reticulum, We localized PL6 to the Golgi using immunofluorescence staining and renamed it Prego (Progesterone REceptor-associated GOlgi protein). We identified the HYA22 gene as a protein phosphatase that regulates the phosphorylation status of the RB TSG thereby negatively controlling the cell cycle and renamed it RBSP. We identified the NPRL2/G21 as a potential novel mismatch repair gene and target of cisplatine. Current/future plans are focused on: (I) Functional analysis of the VHL gene and VHL target genes, specifically: (1) to identify the cis elements and trans factors protecting the VHL CpG promoter. (2) further study the expression and role of carbon